Title: X-ray Absorption Spectroscopy Reveals an Organometallic Ni–C Bond in the CO-Treated Form of Acetyl-CoA Synthase

Acetyl-CoA synthase (ACS) is a key enzyme in the Wood–Ljungdahl pathway of anaerobic CO 2 fixation, which has long been proposed to operate by a novel mechanism involving a series of protein-bound organometallic (Ni–CO, methyl–Ni, and acetyl–Ni) intermediates. Here we report the first direct structural evidence of the proposed metal–carbon bond. We describe the preparation of the highly active metal-replete enzyme and near-quantitative generation of the kinetically competent carbonylated intermediate. This advance has allowed a combination of Ni and Fe K-edge X-ray absorption spectroscopy and extended X-ray absorption fine structure experiments along with density functional theory calculations. The data reveal that CO binds to the proximal Ni of the six-metal metallocenter at the active site and undergoes dramatic structural and electronic perturbation in forming this organometallic Ni–CO intermediate. This direct identification of a Ni–carbon bond in the catalytically competent CO-bound form of the In conclusion, a cluster of ACS provides definitive experimental structural evidence supporting the proposed organometallic mechanism of anaerobic acetyl-CoA synthesis.